Listeria monocytogenes is facultative intracellular food-borne pathogen that provides an extremely amenable model for basic studies on host-pathogen interactions. A primary determinant of L. monocytogenes pathogenesis is Listeriolysin O (LLO), a member of a large family of pore-forming cytolysins that is largely responsible for mediating escape of L. monocytogenes from a phagosome and for virulence. LLO activity is a double-edged sword as its activity must be restricted to an acidic phagosome or the host cell will die due to LLO-mediated cell death (referred to as compartmentalization). In contrast, the ActA protein is only expressed by bacteria when they reach the host cytosol. Despite the fact that LLO and ActA act in different compartments and at different times during the infection cycle, the transcription of both is entirely dependent on the same Crp family transcription factor PrfA. PrfA is predicted to bind an activating ligand, but the nature of the ligand is not known. During the prior funding period, we developed innovative tools and performed extensive genetic screens to identify L. monocytogenes determinants that regulate compartment-specific expression of LLO and ActA. The results of the screens implicate a critical role of redox stress as a mediator of compartmentalization. L. monocytogenes mutants lacking glutathione synthase (GshF) expressed very low levels of ActA during cytosolic growth and were greater than 1000-fold less virulent in mice. These data suggest that cytosolic L. monocytogenes are experiencing redox stress, but of unknown origin. However, the defect in ActA expression was rescued by a mutation that locks PrfA in its activated state, strongly suggesting that a primary role for glutathione is to mediate PrfA activation and we hypothesize that glutathionylation is the activating ligand. We also found that LLO is glutathionylated. We hypothesize that glutathionylation protects the LLO cysteine from irreversible oxidation in a phagosome prevents its activity in the cytosol. We propose to test the role of glutathione on LLO activity and compartmentalization, evaluate the role played by glutathione in PrfA activation and test the hypothesis that PrfA is a sensor of phagosomal and cytosolic redox stress.